Proteome Discoverer 1.4/2.0/2.1/ and 2.2(!!) Videos

This is the complete list of videos for Thermo Scientific's Proteome Discoverer version compiled by me and some others over the years.  As always, please watch all videos in HD for best resolution. Sorry about the sound and my squeaky voice.


New in PD 1.4:
FASTA databases:
Configuring Mascot:
Getting started with the PD 1.4 Daemon:
Setting up/using spectral libraries:
Setting up a simple workflow:
False discovery (PSM validation) nodes:
Workflow templates (exporting/importing):
Using the annotator node:
Running an iTRAQ/TMT experiment:
Processing SILAC data:
Label free quan experiments (PIAD):
Processing neutral loss triggered MS2 LTQ data:
Processing neutral loss triggered MS2 Q Exactive data:
Exporting unmatched spectra for de novo or PTM analysis:
Installing MSAmanda:
Multi-enzyme processing in PD 1.4:


1.       Loading a FASTA database from your hard drive:
2.       Creating a custom FASTA database:
3.       How to use the Administration menu:
4.       How to configure your Mascot server:
5.       How to set up a simple peptide ID workflow in PD 2.0:
6.       How to use the consensus steps in PD 2.0:
7.       How to process M2 TMT and iTRAQ MS2 data:
8.       How to organized a reporter quan experiment in PD 2.0 (Part I):
10.   How to organize a reporter quan experiment in PD 2.0 (Part 2, the results):
11.   How to process SILAC data:
12.   Reprocess consensus report data in PD 2.0 (new version) :
13.   How to perform label free relative protein quan in Proteome Discoverer 2.0:
14.   How to set up a complex relative label free quan experiment in PD 2.0 (multiple fractions):
15.   How to use the Annotation nodes:
16.   How to use the Proteome Discoverer 2.0 Daemon:
17.   Set up a search with multiple search engines:
18.   How to export data to search in another program:
19.   Peptide FDR discussion (PSM validation nodes):

20.   Protein Validation nodes:

21.  How to extract only proteins with a specific post-translational modification:
22.  How to process global peptide and global prosphoproteomics data together into one single report:


Setting up Proteome Discoverer 2.2 for first use:
Adding FASTA files from Hard Drive (from my PD 2.0 videos):
How to configure your Mascot Server (from my PD 2.0 videos):
What is the SpectrumRC node and how do you use it?
Getting started with label free quan with Minora!
A more in-depth look at the precursor ion quantification nodes:
Looking at the Minora results:
Finding what is significantly different in your data with Volcano plots: (Updated Link)
Reporter ion quantification report output:
Using Filters and setting default filters for PD 2.2:
Optimizing the MSF Files consensus node:


  1. I have question regarding Label free quan experiments (PIAD): You have selected precurssor ion detector in the quantification experiment. However there is one more option which precursor ion quantifier. Can you please share your thoughts on this?

  2. Santosh,
    There are two quan settings with surprisingly similar names. The "area" detector is for label free quan. The other one is the SILAC type experiment. Does this clarify?

  3. Ben. Yes clear now. Thanks for the prompt reply..

  4. Hey Ben, Few more questions on label free quan using PD 1.4. If i have lets say 4 technical replicates of my sample, so after uploading the raw files in a workflow template. Whether the PD will merge my tech replicate and will give combined result of that particular sample? and the other question is, is it possible to do LFQ of several case control pairs with replicates at a time on PD 1.4

  5. Hi Ben,

    Do you have anything on setting up a very basic Workflow for
    an Orbitrap XL using only CID, with 1 hi-res Orbitrap scan
    followed by 8 linear trap MS/MS scans ????

    And all of the recommended parameters ?



    BTW: Great Job on thee.
    Best PD Resource out there, bar NONE !!!

    1. Mark,
      Thanks! I'm glad you find this useful. I should have exactly that method floating around here somewhere from my postdoc. Can you send me a request to so I have a reminder and your email address? I'll try and get it to you in the next day or so.

  6. Hi Ben,

    I am wondering how I can do labeling methods if i have PD 1.3 and not 1.4. What can i do or use in such case? Thanks for your help

    1. Hi,
      Absolutely! If you have the quan version of PD, everything from 1.1 (I think...defnitely 1.2) can do labeled quantification.
      But, unless there is a definite reason you have to stay on PD 1.3, the upgrade to PD 1.4, in most cases, is free. And it is substantially better.

  7. Hi Ben,

    I have used "area detector" node to perform label-free quantification on PD 1.4. I noticed that protein areas may change depending on the manner I deal with my data. Working with triplicates I can select the three .raw files and perform only one search, however, it is possible to search them separately and open a unique report sheet merging the three independent runs. I am wondering what's the best way to do that, could you give an advice?

  8. Hi Ben,
    Thank you for put together all these tools are very useful. I am quite new at iTRAQ proteomics and I'm using PD1.4. I have been struggling fpr a while dealing with missing quan values. I am comparing a control group (3 tags) and a diseased one (4 tags) (one tag for a master pool), and we are very interested in the proteins that are exclusively expressed for the diseased group, but the ratios of these proteins are not reported as you can suppose. Could you recommend a strategy to find this specific group of proteins? Any help is greatly appreciated.